Abstract 641: Identification of anti-tumoral neutrophil phenotypes induced by immunotherapies in head and neck cancer

Abstract Only ∼20% of recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) patients respond to immune-checkpoint blockade (ICB) therapy. A major roadblock is the immunosuppressive nature of the tumor microenvironment. Increased infiltration of neutrophils in blood and tumors of HNSCC patients is associated with worse prognosis and ICB therapy resistance. Depleting neutrophils, however, with or without ICB treatment, has not inhibited tumor growth in mice bearing syngeneic MOC2 tumors, which are phenotypically analogous to immunologically “cold”, aggressive HNSCC tumors. We hypothesize that this is due to the heterogeneity of tumor-infiltrated neutrophils with both pro- and anti-tumoral phenotypes. This study uses multi-omics single-cell profiling, combining RNA and protein, to identify pro- and anti-tumoral neutrophil states in MOC2 tumors treated with recently reported neutrophil-related therapies. Specifically, we tested anti-CD40 agonist, TNF-alpha, and both in combination with cetuximab, an anti-tumor antibody (designated Neutrophil Activating Therapy, NAT) that has been recently reported in other cancers. Cetuximab (an anti-EGFR mAb) was used for MOC2-huEGFR (M2h), an HNSCC cancer model transduced with human-EGFR. We found this treatment induced new neutrophil subsets, defined by scRNA-Seq bioinformatics analysis. One subset has recently reported interferon-stimulated phenotypes, which we found in the anti-CD40 alone, and NAT combinatorial treatment groups; these two groups also showed the best anti-tumor efficacy. Gene signature of this subset is also found in scRNA-Seq of infiltrating neutrophils in our previous K17-KO MOC2 model, which shows spontaneous tumor regression in the absence of therapy. In addition, we identified a neutrophil subset associated with migration-related transcription factors only in NAT and TNFa treatment groups, which might explain why NAT is superior to anti-CD40 alone. Our findings thus far demonstrate the presence of neutrophil heterogeneity, with some phenotypes associated with anti-tumor responses, including a potentially new phenotype, in a murine head and neck cancer model induced by a combination of immunotherapies. Ongoing and future work includes identifying specific synergistic mechanisms of the anti-tumoral neutrophil subpopulations induced by anti-CD40 and TNF-alpha treatment and if these phenotypes are also seen in ICB-treated HNSCC patients who respond to their therapy. We anticipate our characterization of anti-tumoral neutrophil phenotypes induced by NAT in M2h tumors may provide insights into how more effective immunotherapy regimens beyond ICB can be designed to confer tumor clearance and prolonged survival in patients with head and neck cancer. Citation Format: Anqi Gao, Amy Erbe-Gurel, Natalie B. Winn, Joel Matthews, Kaia N. Heimstreet, Wei Wang, Paul F. Lambert, Paul M. Sondel, Huy Q. Dinh. Identification of anti-tumoral neutrophil phenotypes induced by immunotherapies in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 641.